The proper assembly of the mitotic spindle is essential for the segregation of genetic material during cell division. Mis-regulation of this process contributes to developmental abnormalities and tumor progression. Recent work from our laboratory has demonstrated a novel role for integrin activity in regulating centrosome function and microtubule assembly. Specifically, we showed that a mutation (Y783A) in the integrin 21 subunit cytoplasmic domain (21 tail) that inhibits integrin activation also prevents the proper assembly of the microtubule cytoskeleton during interphase, the formation of a bipolar spindle at mitosis, and cytokinesis. Importantly, experimentally activating the mutant integrin restores microtubule assembly both during interphase and mitosis and promotes normal cell division. Our long-term goal is to understand the molecular mechanisms by which integrins regulate these fundamental processes. This application proposes studies aimed at understanding how defects in integrin signaling inhibit centrosome function and microtubule assembly during interphase, since defects at this point of the cell cycle could contribute to spindle defects at mitosis. Preliminary studies demonstrate that adhesion-induced FAK activation, Src signaling and the formation of focal adhesions are inhibited in cells adhered by the mutant integrin. We have also identified PKC4 as an important regulator of microtubule assembly and our preliminary data suggest that the centrosomal localization of PKC4 may be important. Based on these findings, we hypothesize that in cells adhered by the Y783A mutant integrin, defects in FAK/Src signaling result in the mis-regulation of Rho family kinases and prevent the activation and/or the centrosomal localization of PKC4, and that the mis-regulation of these pathways results in the inhibition of MT nucleation and growth. We will test these hypotheses using molecular genetic approaches to alter the activation of signaling pathways, the centrosomal localization of PKC4 and the formation of focal adhesions in cells adhered by the wild-type and mutant integrin. Microtubule assembly and focal adhesion formation will be analyzed by immunofluorescence microscopy of fixed cells. The rate of microtubule nucleation and microtubule dynamics will be analyzed by real-time microscopy. The completion of these studies will provide mechanistic insight into how integrins regulate centrosome function and microtubule assembly during interphase, which will provide an important foundation for understanding how integrins regulate spindle assembly and cytokinesis. PUBLIC HEALTH RELEVANCE: The proposed studies are directed at understanding how cells regulate the assembly of their microtubule cytoskeleton. This is a fundamental cellular process important for normal cellular physiology, the migration of cells and the proper segregation of genetic material during development. The mis-regulation of this process contributes to tumor metastasis and errors in the segregation of genetic material contributing to the formation of cancer cells and other genetic defects.